Regimen for suppressing organ rejection

ABSTRACT

The present invention relates to a method of suppressing organ rejection in a patient receiving an organ transplant by initiating oral treatment with a once-daily extended release tacrolimus dosage form, for example, at an initial dose of from about 0.15 to about 0.20 mg/kg/day within 24 or 48 hours following transplantation. The once-daily extended release tacrolimus dosage form (i) provides low fluctuation and/or swing of tacrolimus, (ii) provides a significantly lower C max  than an immediate release formulation of tacrolimus while providing the same or greater area under the curve (AUC), (iii) releases the tacrolimus substantially in the colon and/or the lower ileum, (iv) releases at most 63.5% of the tacrolimus in the dosage form at the 12 hour time point, or (v) any combination of any of the foregoing.

This application claims priority to U.S. Provisional Application No.61/840,354, filed Jun. 27, 2013, which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to a method of suppressing organ rejectionin a patient receiving an organ transplant (e.g., a de novo kidneytransplant recipient) by initiating oral treatment with a once-dailyextended release tacrolimus dosage form, for example, at an initial doseof from about 0.15 to about 0.20 mg/kg/day, within 24 or 48 hoursfollowing transplantation. The once-daily extended release tacrolimusdosage form (i) provides low fluctuation and/or swing of tacrolimus,(ii) provides a significantly lower C_(max) than an immediate releaseformulation of tacrolimus while providing the same or greater area underthe curve (AUC), (iii) releases the tacrolimus substantially in thecolon and/or the lower ileum, (iv) releases at most 63.5% of thetacrolimus in the dosage form at the 12 hour time point, or (v) anycombination of any of the foregoing.

BACKGROUND OF THE INVENTION

Tacrolimus is a macrolide lactone also known as FK506, fugimycin ortsukubaenolide. Tacrolimus is marketed under the tradenames Prograf® andAdvagraf® as an immunosuppressive agent to prevent allograft rejection,i.e. rejection of transplanted organs. In the U.S., Prograf® (NDA No.050708) is approved for the prophylaxis of organ rejection in patientsreceiving allogeneic liver, kidney, or heart transplants

Advagraf is a once-daily formulation which has not received approval inthe U.S. Advagraf failed a non-inferiority study a few years ago asreported in Srinivas et al., Am. J. Transplant 2010; 10: 2571-2573.According to Srinivas,

-   -   Although, it has never been definitively determined whether        calcineurin inhibitor (CNI) efficacy in general and tacrolimus        efficacy in particular is best correlated with AUC, peak or        trough, if the AUCs were similar in this study the obvious        conclusion would be that the observed efficacy failure [of        Advagraf] is most likely related to lower and less frequent        peaks with the once a day formulation. Interestingly, in the        other phase III trial with once a day tacrolimus, rejection        rates were also higher compared to twice a day tacrolimus with        equivalent trough levels throughout the study, a further        indication that the lower and less frequent tacrolimus peak        might be detrimental to efficacy

Similarly, Barraclough et al., Drugs 2011, 71(12): 1561-1577, reported:

-   -   Although efficacy of tacrolimus has never been definitively        associated with peak or trough concentrations or AUC, an        association of peak ciclosporin concentrations with freedom from        acute rejection has been demonstrated. This suggests the        possibility that the higher rejection rates observed in the        previously mentioned studies may be related to the lower and        less frequent concentration peaks that are seen with the        Advagraf® formulation.

WO 2005/020993, WO 2005/020994, WO 2008/0145143, WO 2010/005980, and WO2011/100975 disclose tacrolimus-containing pharmaceutical compositionswith improved bioavailability.

There is a continuing need for an improved once-daily tacrolimus regimenwhich has the same or better efficacy than twice-daily tacrolimus withfewer adverse events.

SUMMARY OF THE INVENTION

The present inventors have discovered that certain once-daily extendedrelease dosage forms are particularly effective at suppressing earlyacute organ rejection (for example, in de novo transplant recipients).The extended release dosage form (i) provides low fluctuation and/orswing of tacrolimus (relative to, for example, Prograf and/or Advagraf),(ii) provides a significantly lower C_(max) (e.g., at least 15% lower(e.g., 15-30% lower C_(max)) than an immediate release formulation oftacrolimus (such as Prograf) at the same dose while providing the sameor greater area under the curve (AUC), (iii) releases the tacrolimussuch that absorption of the tacrolimus takes place substantially in thecolon (such as one or more locations of the colon ascendens, colontransversum and colon decendens) and/or the lower ileum, (iv) releasesat most 63.5% of the tacrolimus in the dosage form at the 12 hour timepoint, or (v) any combination of any of the foregoing.

The extended release dosage forms permit a higher initial daily dose oftacrolimus to be administered to the transplant recipient (i.e., atransplant patient who has not previously received tacrolimus followingthe transplantion), without a significant increase in adverse events.Without being bound by any particular theory, it is theorized that thehigher initial daily dose results in improved imunosuppression in thetransplant recipient which in turn reduces the risk of acute rejectionof the organ following transplantation. In one embodiment, the initialdaily dose of tacrolimus is from about 0.15 to about 0.20 mg/kg, e.g.,about 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 mg/kg. In a preferredembodiment, the initial daily dose of tacrolimus is about 0.17 mg/kg. Inanother embodiment, the transplant recipient has never previously beenadministered tacrolimus. In yet another embodiment, treatment with theextended release tacrolimus dosage form is initiated within 24 or 48hours of receiving the organ transplant.

One embodiment of the present invention is a method of suppressing organrejection in a recipient of a liver, kidney, or heart transplant (e.g.,an allogeneic liver, kidney, or heart transplant). The method includesinitiating oral treatment with a once-daily extended release tacrolimusdosage form of the present invention at an initial dose of from about0.15 to about 0.20 mg/kg/day within 24 or 48 hours followingtransplantation. The extended release tacrolimus dosage form preferablyhas a lower fluctuation and/or swing than Prograf and/or Advagraf. Forexample, the extended release tacrolimus dosage form preferably has afluctuation of less than 100%, a swing less than 120%, or both.

Another embodiment is a method of suppressing organ rejection in a denovo transplant recipient (e.g., a de novo kidney transplant patient) by

-   -   (a) initiating oral treatment with a once-daily extended release        tacrolimus dosage form as described herein at an initial dose of        from about 0.15 to about 0.20 mg/kg within 24 or 48 hours        following transplantation;    -   (b) concomitantly treating the recipient with an IL-2 receptor        antagonist (such as basiliximab) and mycophenolate mofetil; and    -   (c) adjusting the dose of the extended release tacrolimus dosage        form so that the whole blood pre-dose (trough) concentration of        tacrolimus is maintained in the range of from about 5 to about        20 ng/mL.

In one embodiment, step (c) includes adjusting the dose of the extendedrelease tacrolimus dosage form so that the whole blood pre-dose (trough)concentration of tacrolimus is maintained in the range of from about 6to about 11 ng/mL for the first 30 days, and from about 4 to about 11ng/mL after 30 days. In another embodiment, step (c) includes adjustingthe dose of the extended release tacrolimus dosage form so that thewhole blood pre-dose (trough) concentration of tacrolimus is maintainedin the range of from about 4 to about 11 ng/ml during the first 3 or 12months post-transplant.

In another embodiment, 1 g of mycophenolate mofetil is administeredtwice daily (2 g daily dose).

In yet another embodiment, a first 20 mg dose of basiliximdab isadministered 2 hours prior to transplantation, and a second 20 mg doseof basiliximdab is administered 4 days after transplantation.

Step (b) may further include also concomitantly treating the recipientwith one or more corticosteroids, such as prednisone or an equivalent toit. In one embodiment, the recipient is maintained for the first monthpost-transplant on a minimum of 10 mg of prednisone or an equivalent toit per day and optionally a minimum of 5 mg of prednisone or anequivalent to it per day thereafter.

Yet another embodiment is a method of suppressing organ rejection in ade novo transplant recipient (e.g., a de novo kidney transplant patient)by

-   -   (a) initiating oral treatment with a once-daily extended release        tacrolimus dosage form as described herein at an initial dose of        from about 0.15 to about 0.20 mg/kg within 24 hours following        transplantation;    -   (b) concomitantly treating the recipient with azathioprine or        other purine biosynthesis inhibitor; and    -   (c) adjusting the dose of the extended release tacrolimus dosage        form so that the whole blood pre-dose (trough) concentration of        tacrolimus is maintained in the range of from about 5 to about        20 ng/mL. In one embodiment, step (c) includes adjusting the        dose of the extended release tacrolimus dosage form so that the        whole blood pre-dose (trough) concentration of tacrolimus is        maintained in the range of from about 7 to about 20 ng/mL for        the first 3 months, and from about 5 to about 15 ng/mL for the        next 9 months.

Yet another embodiment is a method of reducing or minimizing the risk orincidence of complications due to organ transplantation (such as kidneytransplantation) by performing any of the method steps discussed herein.

The method steps of the present invention also result in reducedincidence of complications of the transplanted organ (e.g., kidney),peripheral oedema, diarrhea, deep vein thrombosis (DVT), constipation,anemia, low blood phosphate, nausea, or any combination of any of theforegoing.

Yet another embodiment is a method of reducing or minimizing the risk orincidence of adverse events in a patient in need of tacrolimus (such asa transplant recipient) by performing any of the method steps discussedherein. The adverse events which can be reduced or minimized includecomplications of the transplanted organ (e.g., kidney), peripheraloedema, diarrhea, deep vein thrombosis (DVT), constipation, anemia, lowblood phosphate, nausea, or any combination of any of the foregoing.

In any of the embodiments described above, the patient can be a kidneytransplant patient such as a de novo kidney transplant patient.

Yet another embodiment is a method of suppressing organ rejection in arecipient of a liver transplant (e.g., an allogeneic liver transplant, ade novo liver transplant patient, or a de novo allogeneic livertransplant). The method includes initiating oral treatment with aonce-daily extended release tacrolimus dosage form of the presentinvention at an initial dose of from about 0.15 to about 0.20 mg/kg/day,or a dose to provide and maintain a whole blood pre-dose (trough)concentration of tacrolimus in the range of from about 5 to about 20ng/mL, within 24 or 48 hours following transplantation.

Yet another embodiment is a method of suppressing organ rejection in arecipient of an allogeneic heart transplant (e.g., an allogeneic hearttransplant, a de novo heart transplant patient, or a de novo allogeneicheart transplant patient). The method includes initiating oral treatmentwith a once-daily extended release tacrolimus dosage form of the presentinvention at an initial dose of from about 0.15 to about 0.20 mg/kg/day,or a dose to provide and maintain a whole blood pre-dose (trough)concentration of tacrolimus in the range of from about 10 to about 20ng/mL, within 24 or 48 hours following transplantation. In oneembodiment, the method further includes adjusting the dose of theextended release tacrolimus dosage form so that the whole blood pre-dose(trough) concentration of tacrolimus is maintained in the range of fromabout 5 to about 20 ng/ml during the first 3 months, and in the range offrom about 5 to about 15 ng/ml after.

The patient in any of the embodiments described above may suffer from,or have a history of, one or more of peripheral oedema, diarrhea, deepvein thrombosis, constipation, anemia, low blood phosphate, and nausea.The patient in any of the embodiments described above may be Caucasianor African-American. Furthermore, the transplant recipient in any of theembodiments described above can be an adult kidney transplant recipient,such as a de novo adult kidney transplant recipient.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “bioequivalency” denotes a scientific basis onwhich generic and brand name drugs are compared with one another. Forexample, drugs are bioequivalent if they enter circulation at the samerate when given in similar doses under similar conditions. Parametersoften used in bioequivalence studies are t_(max), C_(max),AUC_(0-infinity), and AUC_(0-t). Furthermore, in the present context, adosage form is regarded as bioequivalent to Prograf® or a similarcommercially available tacrolimus-containing product if the value of theparameter (e.g., C_(max) and/or AUC) used is within 80-125% of that ofPrograf® or the similar commercially available tacrolimus-containingproduct used in the test.

In the present context “t_(max)” denotes the time to reach the maximalplasma concentration (C_(max)) after administration. AUC_(0-infinity)denotes the area under the plasma concentration versus time curve fromtime 0 to infinity. AUC_(0-t) denotes the area under the plasmaconcentration versus time curve from time 0 to time t. MRT denotes meanresidence time for tacrolimus. Swing denotes (C_(max)−C_(min))/C_(min)and fluctuation denotes (C_(max)−C_(min))/C_(average). Peak-troughfluctuation denotes C_(max)/C_(min).

Unless otherwise specified, Prograf refers to the drug product approvedby the U.S. Food and Drug Administration under New Drug Application(NDA) No. 050708.

Unless otherwise specified, Advagraf refers to the drug product approvedby the European Medicines Agency under EMA Product No. EMEA/H/C/000712.

Dosage Regimen

In one embodiment, the initial daily dose of tacrolimus is from about0.15 to about 0.20 mg/kg, e.g., about 0.15, 0.16, 0.17, 0.18, 0.19, or0.20 mg/kg. In another embodiment, the initial daily dose of tacrolimusis about 0.17 mg/kg. In another embodiment, the transplant recipient hasnever previously been administered tacrolimus. In yet anotherembodiment, treatment with the extended release tacrolimus dosage formis initiated within 24 or 48 hours of receiving the organ transplant. Inyet another embodiment, the patient is Caucasian.

Many African-American patients have more rapid clearance of tacrolimus(for example due to the expression of the Cyp 3A5*1 genotype). As aresult, the dose for African-American patients may be higher than thosefor other patients. For example, the dose for African-American patientsmay range from about 0.20 to about 0.30 mg/kg, such as from about 0.21mg/kg to about 0.27 mg/kg.

The extended release oral dosage form may be administered in the morningor evening (e.g., at bedtime). In one embodiment, the dosage form isadministered in the morning each day. In another embodiment, the dosageform is administered in the evening each day.

Another embodiment is a method of suppressing organ rejection in a denovo kidney transplant recipient by

-   -   (a) initiating oral treatment with a once-daily extended release        tacrolimus dosage form as described herein at an initial dose of        from about 0.15 to about 0.20 mg/kg within 24 or 48 hours        following transplantation;    -   (b) concomitantly treating the recipient with an IL-2 receptor        antagonist (such as basiliximab) and mycophenolate mofetil; and    -   (c) adjusting the dose of the extended release tacrolimus dosage        form so that the whole blood pre-dose (trough) concentration of        tacrolimus is maintained in the range of from about 5 to about        20 ng/mL.

In one embodiment, step (c) includes adjusting the dose of the extendedrelease tacrolimus dosage form so that the whole blood pre-dose (trough)concentration of tacrolimus is maintained in the range of from about 6to about 11 ng/mL for the first 30 days post-transplant, and from about4 to about 11 ng/mL after 30 days.

In another embodiment, step (c) includes adjusting as necessary the thedose of the extended release tacrolimus dosage form(s) so that the wholeblood pre-dose (trough) concentration of tacrolimus is maintained in therange of from about 8.8 to about 9.3 ng/mL from day 2 to week 3 oftacrolimus treatment, and from about 6.5 to about 8.8 ng/mL for month 1to 12.

In another embodiment, 1 g of mycophenolate mofetil is administeredtwice daily (2 g daily dose).

In yet another embodiment, a first 20 mg dose of basiliximdab isadministered 2 hours prior to transplantation, and a second 20 mg doseof basiliximdab is administered 4 days after transplantation.

Step (b) may further include also concomitantly treating the recipientwith one or more corticosteroids, such as prednisone or an equivalent toit. In one embodiment, the recipient is maintained for the first monthpost-transplant on a minimum of 10 mg of prednisone or an equivalent toit per day and optionally a minimum of 5 mg of prednisone or anequivalent to it per day thereafter.

In another embodiment, the patient's tacrolimus blood concentrations aremaintained between about 7 and about 20 ng/ml. For example, thepatient's tacrolimus blood concentrations can be maintained betweenabout 7 and about 20 ng/ml for the first 3 months (months 1-3) (e.g.,approximately 7 to 12 ng/ml at steady state, e.g., after 7 days oftreatment), and between about 5 and about 15 ng/ml for the next 9 months(months 4-12) (e.g., approximately 7 to 12 ng/ml).

In yet another embodiment, the patient's tacrolimus blood troughconcentrations are maintained between about 7 and about 20 ng/ml. Forexample, the patient's tacrolimus blood trough concentrations can bemaintained between about 7 and about 20 ng/ml for the first 3 months(months 1-3) (e.g., approximately 7 to 12 ng/ml at steady state, e.g.,after 7 days of treatment), and between about 5 and about 15 ng/ml forthe next 9 months (months 4-12) (e.g., approximately 7 to 12 ng/ml).

Organ transplant patients can be converted from a different tacrolimusformulation, such as twice-daily Prograf, another immediate releaseformulation of tacrolimus, Advagraf, or a bioequivalent formulation toone of them, to the extended release dosage form of the presentinvention. Because the extended release dosage form may have improvedbioavailability, the tacrolimus daily dose administered to a patientswitching from Prograf or Advagraf can be lowered, e.g., at a ratio ofabout 0.66-0.80:1 such as about 0.7:1 (extended release dosage form toPrograf or Advagraf). In one embodiment, the conversion is to beperformed at a dosage ratio of about 0.66-0.80:1 (according to theclosest available tablet strength). In other words, for every 1 mg ofPrograf® administered, only about 0.66 to about 0.80 mg of tacrolimus inthe extended release oral dosage form is administered. ForAfrican-American patients switching, the dosage ratio can be about0.85:1. In another embodiment, a patient is switched from Advagraf tothe extended release dosage form of the present invention at a dosageratio of about 0.30-0.75:1 (such as about 0.33-0.7:1).

Transplant patients being treating with cyclosporin instead oftacrolimus, can be converted to the extended release tacrolimus dosageform. The initial dose can be that discussed above (e.g., about 0.15 toabout 0.20 mg/kg). Tacrolimus therapy can be initiated 12 to 24 hoursafter discontinuation of cyclosporin.

In one preferred embodiment, the once-daily extended release tacrolimusdosage form(s) described herein are administered once daily at the sametime, consistently, with or without food. The extended releasetacrolimus dosage form is preferably not taken with an alcoholicbeverage or grapefruit juice and is not chewed, divided, or crushed.

Extended Release Oral Dosage Form

Dissolution and Pharmacokinetics

The oral dosage form releases the tacrolimus over an extended period oftime, for example, over a period of at least 12, 13, 14, 15, 18, 20, 22,or 24 hours. For example, the release may be sufficiently slow that morethan half of the tacrolimus in the dosage form is absorbed in the lowergastrointestinal tract. The release may be sufficiently slow to enable avery low absorption rate whereby its C_(max) is lower than that for animmediate release formulation (such as Prograf) and the minimumconcentration (before the next once daily dose is taken) is greater thanthat for a twice-daily immediate release formulation securing efficacyof the treatment for the full dosing interval of 24 hours.

The fluctuation, peak-trough fluctuation, and/or swing of tacrolimus(total and/or free) blood concentrations (from time 0 to 24 hours)resulting from a single dose of the extended release oral dosage form issignificantly less than that for Prograf. In one embodiment, thefluctuation resulting from the extended release dosage form is less than110, 100, 90, 80, 75, 70, 60, or 50%. In another embodiment, the swingresulting from the extended release dosage form is less than 120, 110,100, 90, 80, 70, or 60%. In yet another embodiment, the peak-troughfluctuation is less than 400, 350, 300, 250, or 200%.

In another embodiment, the fluctuation and/or swing of tacrolimus (totaland/or free) blood concentrations (from time 0 to 24 hours) resultingfrom the extended release dosage form when administered once daily insteady state (e.g., after 7 days of once daily administration) issignificantly less than that for Prograf. In one embodiment, thefluctuation resulting from the extended release dosage form whenadministered once daily in steady state (e.g., after 7 days of oncedaily administration) is less than 100, 90, 80, 75, 70, 60, or 50%. Inanother embodiment, the swing resulting from the extended release dosageform when administered once daily in steady state (e.g., after 7 days ofonce daily administration) is less than 120, 110, 100, 90, 80, 70, or60%.

In yet another embodiment, the fluctuation, peak-trough fluctuation,and/or swing of the blood concentrations, as measured after a singledose or in steady state (e.g., after 7 days of once dailyadministration) is less than that observed for Prograf® and/orAdvagraf®. The decrease is preferably at least 10%, such as at least20%, 30%, 40%, or 50%.

The dosage form upon oral administration may exhibit a C_(max) that issignificantly lower than that of an immediate release tacrolimusformulation such as Prograf®, either after a single dose or at steadystate. In one embodiment, the extended release dosage form exhibits aC_(max) that is at most about 80% of that for an immediate releasetacrolimus formulation (e.g., Prograf®) (e.g., at most 75, 70, 65, 60,55, or 50% of that of) Prograf° when measured after administration of asingle dose. In another embodiment, the extended release dosage formexhibits a C_(max) that is at most about 80% of that for an immediaterelease tacrolimus formulation (e.g., Prograf®) (e.g., at most 75, 70,65, 60, 55, or 50% of that of Prograf®) when measured afteradministration at steady state. In yet another embodiment, the extendedrelease dosage form exhibits a C_(max) that is at most about 80% of thatfor Advagraf ®, e.g., at most 75, 70, 65, 60, 55, or 50% of that ofAdvagraf® when measured after administration of a single dose. In yetanother embodiment, the extended release dosage form exhibits a C_(max)that is at most about 80% of that for Advagraf®, e.g., at most 75, 70,65, 60, 55, or 50% of that of Advagraf® when measured afteradministration at steady state.

The dosage form, when administered at steady state, may provide at_(max) of from about 4 to 8 hours, such as about 5 to 7 hours orapproximately 6 hours.

The dosage form upon oral administration may release the tacrolimus suchthat absorption of the tacrolimus takes place substantially in the colon(such as one or more locations of the colon ascendens, colon transversumand colon decendens) and/or the lower ileum. For example, in oneembodiment, the dosage form releases at least 50, 60, 70, 80, or 90% ofthe tacrolimus in the lower gastrointestinal tract, such as the colon.In another embodiment, the dosage form releases at least 50, 60, 70, 80,or 90% of the tacrolimus in colon ascendens, colon transversum, colondecendens, or any combination of any of the foregoing.

The dissolution profile for the extended release oral dosage form ispreferably measured by USP II dissolution test (paddle) method in 900 mlof an aqueous medium at pH 4.5 (adjusted with 2 N acetic acid) andcomprising 0.005% hydroxypropylcellulose and 0.5% of the surfactantsodium lauryl sulfate (SLS), at 37° C. ±0.5° C. and a paddle speed of100 rpm.

Alternatively, the dissolution profile for the extended release oraldosage form can be measured by USP II dissolution test (paddle) or USP Idissolution test (basket) method in a medium at pH 4.5 and comprising0.005% hydroxypropylcellulose, and a rotation of 50 rpm.

In yet another embodiment, the dissolution profile can be measured byJapanese Pharmacopoeia (13^(th) Ed.), Dissolution Test, No. #2 (Paddlemethod, 50 rpm), using a test solution which is an aqueous 0.005%hydroxypropyl cellulose solution adjusted to pH 4.5. In one embodiment,the dosage form releases less than 63.2% of the tacrolimus in the dosageform at 15 hours as measured by Japanese Pharmacopoeia (13^(th) Ed.),Dissolution Test, No. #2 (Paddle method, 50 rpm), using a test solutionwhich is an aqueous 0.005% hydroxypropyl cellulose solution adjusted topH 4.5.

In one embodiment, the dosage form releases at most 63.5% of thetacrolimus in the dosage form at the 12 hours time point, when testedaccording to USP II dissolution test (paddle) method in 900 ml of anaqueous medium at pH 4.5 and comprising 0.005% hydroxypropylcelluloseand 0.5% of the surfactant sodium lauryl sulfate, at 37° C.±0.5° C. anda paddle speed of 100 rpm. In another embodiment, at most 63.5% of thetacrolimus is released at the 13 time hour point, 14 time hour point, or15 time hour point. In yet another embodiment, the dosage form releasesat least about 50% w/w of the total amount of tacrolimus within about 24hours, such as, e.g., within about 22 hours, within about 20 hours,within about 18 hours, within about 15 hours or within about 12 hours.In yet another embodiment, 63.5% of the tacrolimus is released within 20hours, such as 18 hours, 16 hours or 15.5 hours.

In yet another embodiment, (i) at most 63.5% of the tacrolimus isreleased at the 12 hour time point, and (ii) at least 8% of thetacrolimus is released at 4 hours and/or at least 15% of the tacrolimusis released at hour 8.

In another embodiment, the extended release oral dosage form provides asubstantially zero order release for a majority of the release. In oneembodiment, the oral dosage form provides a substantially zero orderrelease of the tacrolimus in the colon and/or the lower ileum. Forexample, the dosage form may provide a substantially zero order releasefrom 8 hours to 15 hours (for example, when tested according to USP IIdissolution test (paddle) method in 900 ml of an aqueous medium at pH4.5 and comprising 0.005% hydroxypropylcellulose and 0.5% of thesurfactant sodium lauryl sulfate, at 37° C.±0.5° C. and a paddle speedof 100 rpm). In another embodiment, the dosage form provides asubstantially zero order release from 2 hours to 10 hours (for example,when tested according to USP II dissolution test (paddle) method in 900ml of an aqueous medium at pH 4.5 and comprising 0.005%hydroxypropylcellulose and 0.5% of the surfactant sodium lauryl sulfate,at 37° C.±0.5° C. and a paddle speed of 100 rpm).

In yet another embodiment, the addition of a surfactant to the releasemedium provides a release rate of the tacrolimus whereby the release ofat most 80% of the tacrolimus is extended for a period of at least 7hours, such as at least 8 hours, such as at least 9 hours, such as atleast 10 hours, such as at least 11 hours, such as at least 12 hourssuch as at least 13 hours when tested in vitro according to USP IIdissolution test (paddle) method in 900 ml of an aqueous medium at pH4.5 and comprising 0.005% hydroxypropylcellulose and 0.5% of thesurfactant sodium lauryl sulfate, at 37° C.±0.5° C. and a paddle speedof 100 rpm.

In one embodiment, 80% of the tacrolimus is released within 24 hours,such as 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17hours, or 16 hours, when tested in vitro according to USP II dissolutiontest (paddle) method in 900 ml of an aqueous medium at pH 4.5 andcomprising 0.005% hydroxypropylcellulose and 0.5% of the surfactantsodium lauryl sulfate, at 37° C.±0.5° C. and a paddle speed of 100 rpm.

In one embodiment, release of the tacrolimus begins within 120 minutessuch as within 90 minutes, such as within 60 minutes after deposition ofthe dosage form in the dissolution apparatus when tested in vitroaccording to USP II dissolution test (paddle) method in 900 ml of anaqueous medium at pH 4.5 and comprising 0.005% hydroxypropylcelluloseand 0.5% of the surfactant sodium lauryl sulfate, at 37° C.±0.5° C. anda paddle speed of 100 rpm.

The dosage form may also have one or more of the following releasecharacteristics:

a) The dosage form releases at most about 20% w/w of the tacrolimuswithin 1 hours, or within 2 hour, or within 3 hours, or within 4 hoursor within 5 hours, when tested in vitro according to USP II dissolutiontest (paddle) method in 900 ml of an aqueous medium at pH 4.5 andcomprising 0.005% hydroxypropylcellulose and 0.5% of the surfactantsodium lauryl sulfate, at 37° C.±0.5° C. and a paddle speed of 100 rpm,or another dissolution test method described herein.

b) The dosage form releases 40% w/w of the tacrolimus within 10 to14hours such as, e.g., within about 11 to13 hours, when tested in vitroaccording to USP II dissolution test (paddle) method in 900 ml of anaqueous medium at pH 4.5 and comprising 0.005% hydroxypropylcelluloseand 0.5% of the surfactant sodium lauryl sulfate, at 37° C.±0.5° C. anda paddle speed of 100 rpm, or another dissolution test method describedherein.

c) The dosage form releases 20% w/w of the total amount of tacrolimusreleased within 6 to10 hours such as, e.g., within about 7 to 9 hours,when tested in vitro according to USP II dissolution test (paddle)method in 900 ml of an aqueous medium at pH 4.5 and comprising 0.005%hydroxypropylcellulose and 0.5% of the surfactant sodium lauryl sulfate,at 37° C.±0.5° C. and a paddle speed of 100 rpm, or another dissolutiontest method described herein.

d) The dosage form releases 50% w/w of the tacrolimus within 13 to 17hours such as, e.g., within about 14 to 16 hours, when tested in vitroaccording to USP II dissolution test (paddle) method in 900 ml of anaqueous medium at pH 4.5 and comprising 0.005% hydroxypropylcelluloseand 0.5% of the surfactant sodium lauryl sulfate, at 37° C.±0.5° C. anda paddle speed of 100 rpm, or another dissolution test method describedherein.

e) The dosage form provides a release profile which is substantiallylinear in the period from 4 to 8 hours defined as a gradient or slopebeing within 25% of the gradient or slope measured at hour 6, such aswithin 15%, preferable within 10%.

f) The dosage form provides a release profile which is substantiallylinear in the period from 6 to 10 hours defined as a gradient or slopebeing within 25% of the gradient or slope measured at hour 8, such aswithin 15%, preferable within 10%.

g) The dosage form provides a release profile which is substantiallylinear in the period from 8 to 12 hours defined as a gradient or slopebeing within 25% of the gradient or slope measured at hour 10, such aswithin 15%, preferable within 10%.

h) The dosage form provides a release profile which is substantiallylinear in the release period from the time point where 20% is releasedto the time point where 50 or 80% is released defined as a gradient orslope at the 50 or 80% time point being within 25% of the gradient orslope measured at the 20% time point.

i) The release extending mechanism of the dosage form is not apermeation controlling coat.

In other embodiments, the following conditions are fulfilled withrespect to in vitro dissolution tests performed by USP II dissolutiontest (paddle) method in 900 ml of an aqueous medium at pH 4.5 andcomprising 0.005% hydroxypropylcellulose and 0.5% of the surfactantsodium lauryl sulfate, at 37° C.±0.5° C. and a paddle speed of 100 rpm.

i) at most about 30% w/w such as, e.g., at most about 25% w/w, at mostabout 20% w/w, at most about 15% w/w or at most about 10% w/w of thetacrolimus is released within 2 hours;

ii) at most about 10% w/w such as, e.g., at most about 7.5% w/w, at mostabout 5% w/w or at most about 2.5% w/w of tacrolimus is released within2 hours;

iii) at most about 60% w/w such as, e.g., at most about 50% w/w, at mostabout 40% w/w or at most about 30% w/w of tacrolimus is released within15 hours such as, e.g., within about 12 hours;

iv) at most about 40% w/w such as, e.g., at most about 30% w/w, at mostabout 25% w/w or at most about 20% w/w of tacrolimus is released within6 hours; and/or

v) at most about 30% w/w such as, e.g., at most about 25% w/w, at mostabout 20% w/w or at most about 15% w/w of tacrolimus is released within4 hours.

The dosage form of the present invention may provide greaterbioavailability than Prograf. In one embodiment, theAUC_(Dosage Form)/AUC_(Prograf®) value is at least about 1.25 such asabout 1.5 or more, about 1.8 or more, about 1.9 or more, about 2.0 ormore.

The dosage form according to the invention may provide and reduce orabolish the need for administration in connection with food intake,which provide for a higher degree of freedom for the recipient of thedosage forms, and consequently the patients acceptance and/or compliancemay be significantly improved. Furthermore, the dosage forms may providea significant reduction in side effects, especially side effect relatedto a high peak concentration (such as, e.g., nephro- and neuro-toxicity(including tremors), diarrhea, constipation, abdominal pain, andnausea).

The dosage form may have a C_(diff)=[C_(max)−C_(t)(t=12 hours)] that isless than that of Prograf® under the same conditions. If C_(diff) forPrograf® is set to 100 then C_(diff) of a dosage form according to theinvention may be 90 or less such as, e.g., about 85 or less, about 80 orless, about 75 or less, about 70 or less, about 65 or less, about 60 orless, about 55 or less, about 50 or less, about 45 or less or about 40or less.

The dosage form may release tacrolimus in a pH dependent or pHindependent manner.

Upon oral administration to a subject, the dosage form may releasetacrolimus in such a manner that a plasma concentration of at leastabout 5 ng/ml such as, e.g., at least about 7.5 ng/mL or at least about10 ng/mL for a time period of at least about 24 hours is obtained. Inone embodiment, the difference between the peak plasma concentration andplasma concentration measured 24 hours after administration is at mostabout 20 ng/mL such as, e.g., at most about 10 ng/ml, at most about 7.5ng/mL or at most about 5 ng/mL. Preferably, the extended release dosageform(s) when administered maintain the subject's whole blood pre-dose(trough) tacrolimus concentration between about 5 and 20 ng/ml, such asfrom about 7 to about 20 ng/ml, from about 5 to about 15 ng/ml, or fromabout 6 to about 14 ng/ml.

In one embodiment, the extended release dosage form exhibits little tono diurnal effect. In a preferred embedment the difference inbioavailability is substantially independent of the time of the day thedosage is administered. This provides the possibility of a once dailydosage regimen at bedtime or in the evening in addition to the normalmorning dosing. For example, according to one embodiment, the extendedrelease dosage form when administered to a subject after at least 4hours fasted state in the evening provides a bioavailability which,relative to that obtained after administration of the dosage form in themorning after at least 4 hours fasted state, is at least 70, 80, 85, 90,or 95% of the value measured after administration in the morning. Inanother embodiment, the dosage form when administered to a subject afterat least 4 hours fasted state in the evening provides a C_(max), whichrelative to that obtained after administration of the dosage form in themorning after at least 4 hours fasted state, is at least 70, 80, 85, 90,or 95% of the value measured after administration in the morning.

Formulation

The extended release oral dosage forms may include a dispersion orsolution of the tacrolimus (e.g., a solid dispersion or solid solution).The tacrolimus may be dispersed or dissolved in a hydrophilic orwater-miscible vehicle. The vehicle preferably has a melting point (orpour point) of at least 20° C. The physical state of the dispersionand/or solution may be determined by using various techniques such asHot Stage Microscopy (HSM), Differential Scanning calorimetry (DSC),Scanning Electron Microscopy (SEM) optionally in combination with EnergyDispersive X-ray (EDX), and X-ray powder diffraction. In one embodiment,the tacrolimus is fully dissolved in the vehicle to form a solidsolution at ambient temperature.

In one embodiment, the concentration of tacrolimus in the vehicle isbetween about 0.01% w/w and about 15% w/w, such as from about 0.1 toabout 10% w/w, from about 0.5 to about 5% w/w, or from about 1 to about4% w/w (based upon 100% total weight of the dosage form). For example,the concentration of tacrolimus can be at most 10, 8, 5, 4, 3, or 2%w/w. The concentration of tacrolimus in the vehicle can be at least0.05, 0.1, 0.5, 0.7, or 1% w/w.

The hydrophilic or water-miscible vehicle to be used according to theinvention is preferably one having a melting point (freezing point orpour point) of at least 20° C., such as at least 30, 40, 50, 52, 55, 59,61 or 65° C.

Examples of useful hydrophilic or water-miscible vehicles to be usedaccording to this invention are selected from the group consisting ofpolyethylene glycols, polyoxyethylene oxides, poloxamers,polyoxyethylene stearates, poly-epsilon caprolactone, polyglycolizedglycerides (such as (i) mixtures of monoesters, diesters and/ortriesters of glycerides of long chain (C₁₂ to C₁₈) fatty acids, (ii) PEG(mono- and/or di) esters of long chain (C₁₂ to C₁₈) fatty acids, and(iii) Gelucire® excipients, e.g. Gelucire® 50/13), and mixtures thereof.Other useful hydrophilic or water-miscible vehicles include, but are notlimited to, polyvinylpyrrolidones, polyvinyl-polyvinylacetate copolymers(PVP-PVA), polyvinyl alcohol (PVA), polymethacrylic polymers (EudragitRS; Eudragit RL, Eudragit NE, Eudragit E), cellulose derivatives(including hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose(HPC), methylcellulose, sodium carboxymethylcellulose, and hydroxyethylcellulose), pectins, cyclodextrins, galactomannans, alginates,carragenates, xanthan gums and mixtures thereof.

Further examples of substances useful as vehicles are:

-   -   i) polyethoxylated fatty acids such as, e.g. fatty acid mono- or        diesters of polyethylene glycol or mixtures thereof such as,        e.g. mono- or diesters of polyethylene glycol with lauric acid,        oleic acid, stearic acid, myristic acid, and ricinoleic acid,        and the polyethylene glycol may be selected from PEG 4, PEG 5,        PEG 6, PEG 7, PEG 8, PEG 9, PEG 10, PEG 12, PEG 15, PEG 20, PEG        25, PEG 30, PEG 32, PEG 40, PEG 45, PEG 50, PEG 55, PEG 100, PEG        200, PEG 400, PEG 600, PEG 800, PEG 1000, PEG 2000, PEG 3000,        PEG 4000, PEG 5000, PEG 6000, PEG 7000, PEG 8000, PEG 9000, PEG        1000, PEG 10,000, PEG 15,000, PEG 20,000, and PEG 35,000;    -   ii) polyethylene glycol glycerol fatty acid esters, i.e. esters        such as the above-mentioned but in the form of glyceryl esters        of the individual fatty acids;    -   iii) glycerol, propylene glycol, ethylene glycol, PEG or        sorbitol esters with e.g. vegetable oils such as hydrogenated        castor oil, almond oil, palm kernel oil, castor oil, apricot        kernel oil, olive oil, peanut oil, or hydrogenated palm kernel;    -   iv) polyglycerized fatty acids such as polyglycerol stearate,        polyglycerol oleate, polyglycerol ricinoleate, and polyglycerol        linoleate;    -   v) propylene glycol fatty acid esters such as propylene glycol        monolaurate and propylene glycol ricinoleate;    -   vi) mono- and diglycerides such as glyceryl monooleate, glyceryl        dioleae, glyceryl mono- and/or dioleate, glyceryl caprylate, and        glyceryl caprate;    -   vii) sterol and sterol derivatives;    -   viii) polyethylene glycol sorbitan fatty acid esters        (PEG-sorbitan fatty acid esters) such as esters of PEG with the        various molecular weights indicated above, and the various        Tween® series;    -   ix) polyethylene glycol alkyl ethers such as PEG oleyl ether and        PEG lauryl ether;    -   x) sugar esters such as sucrose monopalmitate and sucrose        monolaurate;    -   xi) polyethylene glycol alkyl phenols such as the Triton® X or N        series;    -   xii) sorbitan fatty acid esters such as the Span® series or        Ariacel® series, e.g. sorbinan monolaurate, sorbitan        monopalmitate, sorbitan monooleate, and sorbitan monostearate;    -   xiii) lower alcohol fatty acid esters such as oleate, isopropyl        myristate, and isopropyl palmitate; and    -   xiv) ionic surfactants including cationic, anionic and        zwitterionic surfactants such as fatty acid salts, bile salts,        phospholipids, phosphoric acid esters, carboxylates, sulfates        and sulfonates.

In one preferred embodiment, the vehicle is a polyethylene glycol (PEG),in particular a PEG having an average molecular weight of at least 1500,preferably at least 3000, more preferably at least 4000, such as atleast 6000. For example, the PEG may have an average molecular weightranging from 1500 to 35000, from 3000 to 35000, from 3000 to 20000, from4000 to 20000, from 3000 to 10000, or from 4000 to 10000. The PEG canbe, for example, PEG 3000, PEG 4000, PEG 6000, or PEG 8000. Thepolyethylene glycol may advantageously be mixed with one or more otherhydrophilic or water-miscible vehicles, for example a poloxamer,preferably in a proportion (on a weight/weight basis) of between 1:3 and10:1, such as between 1:1 and 5:1, between 3:2 and 4:1, between 2:1 and3:1, or about 7:3. A specific example of a useful mixture is a mixtureof PEG6000 and poloxamer 188 in the ratio of 7:3.

Suitable poloxamers (also known as polyoxypropylene-polyoxyethyleneblock copolymers) include, but are not limited to, poloxamer 188,poloxamer 237, poloxamer 338, poloxamer 407, and other block copolymersof ethylene oxide and propylene oxide such as the Pluronic® and/orTetronic® series. Suitable block copolymers of the Pluronic® seriesinclude polymers having a molecular weight of about 3,000 or more suchas, e.g. from about 4,000 to about 20,000 and/or a viscosity(Brookfield) from about 200 to about 4,000 cps such as, e.g., from about250 to about 3,000 cps. Suitable examples include Pluronic® F38, P65,P68LF, P75, F77, P84, P85, F87, F88, F98, P103, P104, P105, F108, P123,F123, F127, 10R8, 17R8, 25R5, and 25R8. Suitable block copolymers of theTetronic® series include polymers having a molecular weight of about8,000 or more such as, e.g., from about 9,000 to about 35,000 and/or aviscosity (Brookfield) of from about 500 to about 45,000 cps such as,e.g., from about 600 to about 40,000. The viscosities given above aredetermined at 60° C. for substances that are pastes at room temperatureand at 77 ° C. for substances that are solids at room temperature. In apreferred embodiment of the present invention, the poloxamer ispoloxamer 188, which has an average molecular weight of about 8400 and amelting point of about 50-54° C.

The extended release oral dosage form may be that described in WO05/020993, US 2006/0287352, US 2010/0105717 or US 2011/0201639, each ofwhich is hereby incorporated by reference in its entirety. In oneembodiment, the extended release oral dosage form is that described inExample 20 of US 2010/0105717.

In another embodiment, the tacrolimus is dissolved or dispersed in ahydrophobic vehicle, such as an oil, an oily material, sorptionmaterial, a wax or a fatty acid derivative, e.g., a wax having a lowmelting point (for example glyceryl monostearate). For instance, thedosage form may include a sorption material selected from silica acid ora derivative or salt thereof including silicates, silicon dioxide(Aeroperl® 300 (available from Degussa, Frankfurt, Germany)) andpolymers thereof, magnesium aluminosilicate and/or magnesiumaluminometasilicate, bentonite, kaolin, magnesium trisilicate,montmorillonite, saponite, and any combination of any of the foregoing.These sorption materials are useful for containing oils or oily-likematerials.

The dosage form may include other excipients, such as modifying releaseagents, fillers, diluents, disintegrants, binders, glidants, lubricants,and any combination of any of the foregoing. Other pharmaceuticallyacceptable excipients which may be included in the dosage form include,but are not limited to, acidifying agents, alkalizing agents,preservatives, stabilizing agents, antioxidants, pH-adjusting agents,buffering agents, chelating agents, coloring agents, complexing agents,emulsifying and/or solubilizing agents, surface-active agents,absorption enhancing agents, flavors and perfumes, taste-masking agents,wetting agents, humectants, sweetening agents, wetting agents, and anycombination of any of the foregoing.

Examples of release modifying agents include, but are not limited to,hydrophilic polymers such as hydroxypropyl cellulose (HPC),hydroxypropyl methyl cellulose (HPMC), methyl cellulose, poly(N-vinyl-2-pyrrolidinone) (PVP), poly(ethylene oxide) (PEO), poly(vinylalcohol) (PVA), xanthan gum, carrageenan, and other such natural andsynthetic materials. A preferred release modifying agent is HPMC.

Examples of suitable fillers, diluents and/or binders include lactose(e.g. spray-dried lactose, α-lactose, β-lactose, Tabletose®, variousgrades of Pharmatose®, Microtose® or Fast-Floc®), microcrystallinecellulose (various grades of Avicel®, Elcema®, Vivacel®, Ming Tai® orSolka-Floc®), hydroxypropylcellulose, L-hydroxypropylcellulose (lowsubstituted), hydroxypropyl methylcellulose (HPMC) (e.g. Methocel E, Fand K, Metolose SH of Shin-Etsu, Ltd, such as, e.g. the 4,000 cps gradesof Methocel E and Metolose 60 SH, the 4,000 cps grades of Methocel F andMetolose 65 SH, the 4,000, 15,000 and 100,000 cps grades of Methocel K;and the 4,000, 15,000, 39,000 and 100,000 grades of Metolose 90 SH),methylcellulose polymers (such as, e.g., Methocel A, Methocel A4C,Methocel A15C, Methocel A4M), hydroxyethylcellulose, sodiumcarboxymethylcellulose, carboxymethylene,carboxymethylhydroxyethylcellulose and other cellulose derivatives,sucrose, agarose, sorbitol, mannitol, dextrins, maltodextrins, starchesor modified starches (including potato starch, maize starch and ricestarch), calcium phosphate (e.g. basic calcium phosphate, calciumhydrogen phosphate, dicalcium phosphate hydrate), calcium sulfate,calcium carbonate, sodium alginate, and collagen.

Examples of diluents include calcium carbonate, dibasic calciumphosphate, tribasic calcium phosphate, calcium sulfate, microcrystallinecellulose, powdered cellulose, dextrans, dextrin, dextrose, fructose,kaolin, lactose, mannitol, sorbitol, starch, pregelatinized starch,sucrose, and sugar.

Examples of disintegrants include alginic acid or alginates,microcrystalline cellulose, hydroxypropyl cellulose and other cellulosederivatives, croscarmellose sodium, crospovidone, polacrillin potassium,sodium starch glycolate, starch, pregelatinized starch, andcarboxymethyl starch (e.g. Primogel® and Explotab®).

Examples of binders include acacia, alginic acid, agar, calciumcarrageenan, sodium carboxymethylcellulose, microcrystalline cellulose,dextrin, ethylcellulose, gelatin, liquid glucose, guar gum,hydroxypropyl methylcellulose, methylcellulose, pectin, PEG, povidone,and pregelatinized starch.

Examples of glidants and lubricants include stearic acid, magnesiumstearate, calcium stearate or other metallic stearate, talc, waxes andglycerides, light mineral oil, PEG, glyceryl behenate, colloidal silica,hydrogenated vegetable oils, corn starch, sodium stearyl fumarate,polyethylene glycols, alkyl sulfates, sodium benzoate, and sodiumacetate.

Examples of antioxidants include ascorbic acid, ascorbyl palmitate,butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorousacid, monothioglycerol, potassium metabisulfite, propyl gallate, sodiumformaldehylde sulfoxylate, sodium metabisulfite, sodium thiosulfate,sulfur dioxide, tocopherol, tocopherol acetate, tocopherolhemisuccinate, and TPGS and other tocopherol derivatives.

The dosage form may, for example, contain an antioxidant and/or astabilizing agent at a concentration ranging from about 0.1% w/w toabout 5% w/w.

When a surfactant or a mixture of surfactants is present in the dosageform, the concentration of the surfactant(s) may range from about0.1-80% w/w, such as from about 0.1 to about 20% w/w, from about 0.1 toabout 15% w/w, from about 0.5 to about 10% w/w, from about 10 to about70% w/w, from about 20 to about 60% w/w or from about 30 to about 50%w/w.

The stabilizing agent may be a pH-regulating pharmaceutical excipient.Preferably, the stabilizing agent is capable of providing a pH below 7in the dosage form, as measured after re-dispersing the dosage form inwater, for example, a pH in the range of 2.5 to 5 or 2.5 to 4 or 3 to3.6 or 3 to 3.5. Suitable stabilizing agents include, but are notlimited to, inorganic acids, inorganic bases, inorganic salts, organicacids, organic bases, and pharmaceutically acceptable salts thereof. Thestabilizing agent can be a chelating agent. For instance, thestabilizing compound can be an organic acid selected from mono-, di-,oligo and polycarboxylic acids, for example succinic acid, citric acid,tartaric acid, acrylic acid, benzoic acid, malic acid, maleic acid,oxalic acid, sorbic acid and mixtures thereof. In one preferredembodiment, the stabilizing agent is oxalic acid, tartaric acid and/orcitric acid. One preferred stabilizing agent is tartaric acid.

The dosage form may include a stabilizing effective amount ofstabilizing agent (for instance, an amount effective to prevent ordecrease the rate of formation of tacrolimus degradation products). Inone embodiment, the amount of stabilizing agent ranges from about 0.05%w/w to about 5% w/w, based upon the total weight of tacrolimus, vehicleand stabilizing agent. The dosage form may contain at least 0.05% w/w,at least 0.1% w/w, or at least 0.2% w/w and less than 3% w/w, less than2% w/w, less than 1% w/w, less than 0.8% w/w, or not more than 0.6% w/wof the stabilizing agent.

Apart from tacrolimus, the dosage form may also comprise a furthertherapeutically, prophylactically and/or diagnostically activesubstance, such as steroids, calcineurin inhibitors and/oranti-proliferative agents. Examples of active substances which can beincluded in the dosage form include prednisone, prednisolone,methylprednisone, cyclosporin, mycophenolate mofetil, azathioprine,sirolimus, everolimus, mycophenolate sodium, and FTY720 (Novartis).

In one embodiment, at least a part of the tacrolimus is present in theform of a solid solution or a solid dispersion. For example, 10% or moresuch as 20, 30, 40, 50, 60, 70, 80, 90, 95% or more, or about 100% w/wof the tacrolimus is present in the dosage form in the form of a solidsolution or a solid dispersion.

A solid dispersion may be obtained, for example, by employing organicsolvents or by dispersing or dissolving the tacrolimus in anothersuitable medium (e.g. an oil or an oily-like material that is in liquidform at room temperature or at elevated temperatures). Solid dispersions(solvent method) may for example be prepared by dissolving a physicalmixture of the tacrolimus and the vehicle (e.g., a hydrophilic polymer)in a common organic solvent, followed by evaporation of the solvent.Suitable organic solvents include, but are not limited to, solvents inwhich the tacrolimus is soluble such as methanol, ethanol, methylenechloride, chloroform, ethylacetate, acetone or mixtures thereof. Thesolid dispersion may also be formed by spray drying techniques,controlled agglomeration, freeze-drying or coating on carrier particlesor any other solvent removal process. For instance, the tacrolimus maybe dispersed and/or dissolved in a vehicle by a controlled agglomerationmethod. Stabilizing agents may be added in order to ensure the stabilityof the solid dispersion or solution.

A preferred method is described in International Publication No. WO03/004001, which is hereby incorporated by reference. The methodcomprises spraying a first composition in liquid form, where the firstcomposition comprises a first vehicle or carrier and having a meltingpoint above 5° C. onto a second composition comprising a second supportor carrier material, the second composition e.g. being in the fluidizedstate and having a temperature below the melting point of the firstvehicle or carrier. The tacrolimus may be present in the first vehicleor carrier composition and/or in the second support or carriercomposition. In one embodiment, the tacrolimus is dissolved in the firstcomposition. The melting point of the carrier or vehicle may be in therange of 10° C. to 150° C., such as 30° C. to 100° C. or 40 ° C. to 50 °C.

The controlled agglomeration method can produce particles of a desirableparticle size. In one embodiment, the particulate material formed bycontrolled agglomeration has a geometric weight mean diameter d_(gw) of≥10 μm, such as ≥20 μm, from about 20 to about 2000 μm, from about 30 toabout 2000 μm, from about 50 to about 2000 μm, from about 60 to about2000 μm, from about 75 to about 2000 μm, from about 100 to about 1500μm, from about 100 to about 1000 μm, from about 100 to about 700 μm, atmost about 400 μ, at most 300 μ, from about 50 to about 400 μm, fromabout 50 to about 350 μm, from about 50 to about 300 μm, from about 50to about 250 μm or from about 100 to about 300 μm.

Polymers containing acidic functional groups may be suitable for soliddispersions, which release the tacrolimus in a preferred pH rangeproviding acceptable absorption in the intestines. Such polymers may beone ore more selected from the group comprising hydroxypropylmethylcellulose phtalate (HMPCP), polyvinyl acetate phtalate (PVAP),hydroxypropylmethylcellulose acetate succinate (HPMCAS), alginate,carbomer, carboxymethylcellulose, methacrylic acid copolymer (EudragitL, Eudragit S), shellac, cellulose acetate phthalate (CAP), starchglycolate, polacrylin, methyl cellulose acetate phtalate,hydroxypropyulcellulose acetate phthalate, cellulose acetateterephtahalate, cellulose acetate isophthalate and cellulose acetatetrimellitate.

The weight ratio of tacrolimus to polymer in the solid dispersion mayrange from about 3:1 to about 1:20, such as from about 3:1 to about 1:5,or from about 1:1 to about 1:3.

The desired release profile of the dosage form may be provided by usingone or more of the following formulation techniques:

i) coating the dosage form with an enteric coating; and/or

ii) incorporating one or more modifying release agents in the dosageform (for example, with the tacrolimus and vehicle).

The modifying release agent can be, for example, included in theextra-granular phase. For example, a solid dispersion or solid solutionof tacrolimus can be prepared as discussed above (e.g., by controlledagglomeration) and the resulting particles can be mixed with anextragranular phase containing a release-modifying agent.

An entero-coated formulation may have the disadvantage of delaying therelease without extending the release and preferably therefore is usedin combination with an extending technology. This type of coating isresistant to release of the tacrolimus until a certain pH is reached.The film alters properties and becomes permeable at a certain pH.Examples of pH-sensitive polymers, which are relatively insoluble andimpermeable at the pH of the stomach, but which are more soluble andpermeable at the pH of the small intestine and colon include, but arenot limited to polyacrylamides, phthalate derivatives such as acidphthalates of carbohydrates, amylose acetate phthalate, celluloseacetate phthalate, other cellulose ester phthalates, cellulose etherphthalates, hydroxypropylcellulose phthalate,hydroxypropylethylcellulose phthalate, hydroxypropylmethylcellulosephthalate, methylcellulose phthalate, polyvinyl acetate phthalate,polyvinyl acetate hydrogen phthalate, sodium cellulose acetatephthalate, starch acid phthalate, styrene-maleic acid dibutyl phthalatecopolymer, styrene-maleic acid polyvinylacetate phthalate copolymer,styrene and maleic acid copolymers, polyacrylic acid derivatives such asacrylic acid and acrylic ester copolymers, polymethacrylic acid andesters thereof, poly acrylic methacrylic acid copolymers, shellac, andvinyl acetate and crotonic acid copolymers. pH-sensitive polymers ofspecific interest include shellac; phthalate derivatives, particularlycellulose acetate phthalate, polyvinylacetate phthalate, andhydroxypropylmethylcellulose phthalate; polyacrylic acid derivatives,particularly polymethyl methacrylate blended with acrylic acid andacrylic ester copolymers; and vinyl acetate and crotonic acidcopolymers.

The release may also be pH independent, e.g., by providing the dosageform with a controlled release coating such as, e.g. a cellulose basedcoating (for example, ethylcellulose) or by providing the composition inthe form of a matrix composition such as, e.g., a hydrophilic cellulosepolymer matrix type e.g. based on HPMC. A combination may also beemployed.

In one embodiment, the dosage form comprises (a) granules of tacrolimusand a hydrophilic vehicle (such as PEG, poloxamer, or a mixture thereof)in the pores of and/or on an inert porous solid carrier (such aslactose, e.g., lactose monohydrate), and (b) one or more releasemodifying agent(s) (e.g., HPMC). For instance, a tablet can be preparedby compressing a mixture of these granules and release modifyingagent(s). The granules can be prepared, for example, by spraying amixture of tacrolimus in a melted hydrophilic vehicle onto the inertsolid carrier. The tacrolimus is preferably dissolved in the meltedvehicle. In one embodiment, the carrier has a d₅₀ less than 200, 100,80, 75, or 50 microns. In another embodiment, the granules have a d₅₀less than 1000, 800, 600, 400, 300, or 250 microns.

In another embodiment, the dosage form is a tablet comprising, byweight, about 30% to about 80% solid dispersion containing tacrolimus,about 15% to about 35% matrix former (such as HPMC), 0% to about 35%lactose, 0% to about 20% microcrystalline cellulose, and about 0.25% toabout 2% lubricant (such as magnesium stearate).

The dosage form preferably contains less than 0.5% w/w of8-epitacrolimus, a degradation product of tacrolimus upon storage (basedupon 100% w/w of total tacrolimus and its degradation products). Also,the dosage form may be substantially free (e.g. contain less than 1,0.5, 0.2, 0.1 or 0.05% w/w) of an organic solvent or organic solventresidues (based upon 100% w/w of the dosage form). In one embodiment,the dosage form comprises less than 0.1, 0.2, or 0.5% of 8-epitacrolimusafter 1, 2, 4, 8, or 12 weeks or 3 or 6 months of storage at 25° C. at60% relative humidity.

The dosage form may be in the form of, for example, tablets, capsulesand sachets.

Suitable dosage forms (strength) range from 0.1 mg to 15 mg oftacrolimus. Preferred strengths for the dosage forms include 0.75, 1,and 4 mg. Other strengths for the dosage form include, but are notlimited to, 2 and 5 mg.

Tacrolimus

The tacrolimus can be in any physical form (amorphous, crystalline form,such as a hydrate or anhydrate, or complex). In one preferredembodiment, the tacrolimus is in amorphous form. In another embodiment,the tacrolimus is tacrolimus monohydrate (shown below). The preparationof tacrolimus is described in, for example, EP-A-0 184 162. Thetacrolimus can have a particle size (d₅₀) in microns or nanometers.

The following examples serve the purpose of illustrating the inventionand are not intended to limiting the scope of the present invention.

EXAMPLE 1 Tacrolimus Extended Release Dosage Form

The 2 mg tacrolimus tablet (referred to as LCP-Tacro) provided below canbe prepared as described in Example 20 of US 2010/0105717, which isherein incorporated by reference.

Extended release composition, stabilized LCP-Tacro 2 mg Tacrolimusmonohydrate (2.00 mg calculated on the 2.0400 mg anhydrous basis)Excipients Butylated hydroxytoluene 10.200 μg Dimethicone 350 0.25500 μgHypromellose 2208 (15,000 cp) 62.866 mg Lactose monohydrate 41.727 mgMagnesium stearate 1.5716 mg Opadry II white 85G18490 4.7232 mgPoloxamer 188 14.688 mg Polyethylene glycol 6,000 34.272 mg Tartaricacid 255.00 μg

Similar 0.75, 1, and 4 mg tacrolimus containing tablets may be prepared.

EXAMPLE 2

A clinical study was performed to evaluate the efficacy and safety ofLCP Tacro (tacrolimus) tablets administered once daily compared toPrograf capsules twice daily as immunosuppression for the prevention ofacute allograft rejection in de novo adult kidney transplant recipientstreated for a 12 month study period followed by a 12 month, blindedextension treatment period.

This was a two-armed parallel group, prospective, randomized,double-blind, double-dummy, multicenter clinical study to establish theefficacy and safety of LCP-Tacro tablets once daily for the preventionof allograft rejection in de novo adult male and female recipients of aprimary or secondary kidney transplant evaluated by a combined efficacyendpoint comprised of acute rejection, graft loss and patient loss.Recipients of a kidney transplant were randomly assigned to once-dailytherapy with LCP-Tacro tablets or to twice-daily therapy with Prografcapsules, each concomitantly administered with mycophenolate mofetil(MMF) and corticosteroids. All patients also received an interleukin-2(IL-2) receptor antagonist (e.g., Simulect®, basiliximab; NovartisPharmaceuticals, East Hanover, N.J.). Following screening,transplantation, and randomization, study visits were conducted over a12-month treatment period, with additional visits during a 12 monthextension period on treatment and a follow-up safety assessment by visitor telephone interview 30 days after withdrawal from study drug.

The de novo patients (18-70 years old) received a primary or secondarykidney transplant from a non-human leukocyte antigen (HLA) identicallive donor or a deceased donor.

LCP-Tacro Dosage: The initial dose of 0.17 mg/kg was administered orallyin the morning (before noon) within 48 hours following transplantation.Subsequent doses were adjusted according to whole blood tacrolimustrough levels. LCP-Tacro tablets were provided in 0.75, 1, and 4 mgdosage strengths.

Prograf Dosage: Starting total daily dose of 0.10 mg/kg was administeredin two equally divided doses, one in the morning (before noon) and onein the evening, per product labeling. The first dose was administered inthe morning (before noon) within 48 hours following transplantation.Subsequent doses were adjusted according to whole blood tacrolimustrough levels. Prograf capules were provided in 0.5, 1, and 5 mg dosagestrengths.

In the initial post-transplant period, whole blood trough levels weremeasured at 24 and 48 hours following the initial dose. The first doseadjustment based on measurement of whole blood tacrolimus levelsgenerally took place 48 hours after the initial dose for both studydrugs. However, if clinically indicated, the dose was adjusted at 24hours post initial dose. Study drugs were adjusted to maintain the wholeblood pre-dose (trough) concentration of tacrolimus in the target rangeof 6-11 ng/mL for the first 30 days, then 4-11 ng/mL for the remainderof the study.

Tacrolimus trough levels were measured at each study visit beginning 24hours after the first dose of study medication. Tacrolimus whole bloodtrough levels were drawn within 30 minutes before the morning dosing.

Placebo: Matching placebos for both the test formulation and referenceformulation were used in the study.

Concomitant Therapy:

(1) Antibody induction therapy: An IL-2 receptor antagonist (e.g.,basiliximab) was administered according to currently approved productlabeling (2 doses of 20 mg each, the first 20 mg dose was given within 2hours prior to transplantation surgery. The recommended second 20 mgdose was given 4 days after transplantation).

Mycophenolate mofetil (MMF) was administered as per the most currentproduct label (1 g administered twice daily (2 g daily dose)).

(2) Corticosteroids: Corticosteroids were required, but the regimen wasbased on the standard of care at the participating site. However, allpatients were maintained for the first month on a minimum of 10 mg and,thereafter, on a minimum of 5 mg of prednisone or equivalent for the360-day duration of the study. During the 12 month treatment extensionperiod, the corticosteroid treatment was based on the standard of careat the participating site.

The use of polyclonal or monoclonal T-cell depleting antibodies,including but not limited to Thymoglobulin® (rabbit antithymocyteglobulin) or Campath® (alemtuzumab) for induction, was not permitted.The use of Rapamune® (rapamycin, sirolimus), Certican® (everolimus) orinvestigational agents was not permitted.

The primary efficacy endpoint for the study was the proportion oftreatment failures within 12 months after randomization to LCP-Tacro. Apatient was considered a treatment failure if the patient experiencedany of the following events during this period: death, graft failure,biopsy-proven acute rejection (BPAR) (Banff grade ≥1A) or lost tofollow-up.

Clinical decisions were based on local biopsy readings. All biopsieswere also read by a central blinded reader and the central reading wasconsidered the definitive data for purposes of the study. The diagnosisand severity assessment of acute rejection was made according to theBanff 2007 criteria.

Various efficacy and safety endpoints produced at 12-month time pointwere summarized for Months 18 and 24 by treatment groups.

After a screening visit and renal transplantation, there were 17scheduled study visits (including randomization visit) during the 12month treatment period and 4 scheduled visits during the extensionperiod, and a follow-up safety telephone interview at Month 25 which was30 days after the end of study drug treatment. Evaluations includedmeasurement of vital signs, complete physical examinations, ECGs,assessments of AEs and concomitant drug use, laboratory tests(hematology, biochemistry [including hepatic, renal and lipid profiles],HbA1c, CMV and BKV and urinalysis), anti-HLA antibodies anddetermination of tacrolimus whole blood trough levels.

Results

The treatment failure results for LCP-Tacro and Prograf are providedbelow. From Month 3 on, the dose of LCP-Tacro was roughly 15% lower thanthe dose of Prograf required to achieve the same therapeutic bloodlevels.

LCP-Tacro Prograf Treatment Failure (N = 268) (N = 275) Treatmentfailure within 12 months for 49 (18.3%) 54 (19.6%) all subjectsAll-cause mortality 8 (3.0%) 8 (2.9%) Graft failure 9 (3.4%) 11 (4.0%)BPAR 35 (13.1%) 37 (13.5%) Lost to follow-up 4 (1.5%) 5 (1.8%) Treatmentfailure within 12 months for 3 (30.0%) 6 (40.0%) Black subjects

The superior efficacy (lower treatment failure rate) of LCP-Tacro isconsistent with a trend found in two prior clinical studies of one-year(or more). In one prior study (n=63), the LCP-Tacro group had atreatment failure rate of 6.3% while that of the Prograf group was 9.7%.In the other prior study (n=326), the LCP-Tacro group had a treatmentfailure rate of 2.5% versus 4.9% for Prograf.

LCP-Tacro also exhibited superior efficacy during the earlypost-transplant period. According to a Kaplan-Meier analysis of the timeto occurrence of first biopsy-proven acute rejection (BPAR) during thefirst three months post-transplant, LCP-Tacro was superior to Prograf.Within the first 3 months after transplant, when patients are at thegreatest risk of rejection, the treatment failure rates for LCP-Tacroand Prograf were 10.4% and 14.2%, respectively (p=0.124). Within thefirst 6 months after transplant, the treatment failure rates forLCP-Tacro and Prograf were 14.2% and 15.3%, respectively. This incontrast to Advagraf which was found in a prior clinical study toexhibit a quantitatively (though not statistically significant) worsetime to occurrence of first BPAR by a similar analysis. Silva et al.,Am. J. Transplant, 2007, 7:595-608 (see FIG. 3). LCP-Tacro also producedfewer clinically suspected and treated rejections than Prograf (13.8%vs. 15.6%). Additionally, LCP-Tacro rapidly attained therapeutic bloodlevels.

The number of certain adverse events also was lower in the LCP-Tacrogroup.

LCP-Tacro Prograf Adverse Events (N = 268) (N = 275) Complications oftransplanted kidney 19 (7.1%) 30 (10.9%) Peripheral oedema 42 (15.7%) 57(20.7%) Diarrhea 82 (30.6%) 92 (33.5%) Deep vein thrombosis 0 (0%) 6(2.2%) Constipation 18.3% 24.4% Anemia 26.1% 28.7% Low blood phosphate13.4% 15.3% Nausea 13.1% 14.9%

All references, including published patent applications and patents, arehereby incorporated by reference.

1. A method of suppressing organ rejection in a patient receiving aliver, kidney, or heart transplant, the method comprising initiatingoral treatment with a once-daily extended release tacrolimus dosage format an initial dose of from about 0.15 to about 0.20 mg/kg/day followingtransplantation, wherein the once-daily extended release tacrolimusformulation provides a fluctuation of less than 100%, a swing less than120%, or both.
 2. A method of suppressing organ rejection in a patientreceiving a liver, kidney, or heart transplant, the method comprisinginitiating oral treatment with a once-daily extended release tacrolimusdosage form at an initial dose of from about 0.15 to about 0.20mg/kg/day following transplantation, wherein the once-daily extendedrelease tacrolimus formulation releases the tacrolimus substantially inthe colon and/or the lower ileum.
 3. The method of claim 1, whereindosing is initiated within 48 hours following transplantation.
 4. Themethod of claim 1, wherein the extended release dosage form releases atleast 50% of the tacrolimus in the colon and/or the lower ileum.
 5. Themethod of claim 4, wherein the extended-release dosage form releases atleast 50% of the tacrolimus in one or more of the colon ascendens, colontransversum and colon decendens. 6-7. (canceled)
 8. The method of claim1, wherein the extended release tacrolimus dosage form is administeredonce daily in the morning.
 9. The method of claim 1, wherein theextended release tacrolimus dosage form is administered without food.10. The method of claim 1, wherein the dose of the extended releasetacrolimus dosage form is not adjusted during the first 48 hours afterthe initial dose. 11-40. (canceled)
 41. A method of suppressing organrejection in a de novo kidney transplant recipient, the methodcomprising initiating oral treatment with a once-daily extended releasetacrolimus dosage form at an initial dose of from about 0.15 to about0.20 mg/kg/day following transplantation, wherein (i) the once-dailyextended release tacrolimus formulation provides a fluctuation of lessthan 80% and or a swing less than 120%; and (ii) the once-daily extendedrelease tacrolimus formulation releases the tacrolimus substantially inthe colon and/or the lower ileum.
 42. The method of claim 41, whereindosing is initiated within 48 hours following transplantation.
 43. Themethod of claim 41, wherein the extended release dosage form releases atleast 50% of the tacrolimus in the colon and/or the lower ileum.
 44. Themethod of claim 41, wherein the extended release dosage form releases atleast 50% of the tacrolimus in one or more of the colon ascendens, colontransversum and colon decendens.
 45. The method of claim 41, wherein thedosage form comprises (a) granules of tacrolimus and a hydrophilicvehicle in the pores of and/or on an inert porous solid carrier, and (b)a release modifying agent.
 46. The method of claim 41, wherein thedosage form is prepared by compressing into a tablet a mixture of (a)granules of tacrolimus and a hydrophilic vehicle in the pores of and/oron an inert porous solid carrier, and (b) a release modifying agent. 47.The method of claim 46, wherein the granules are prepared by spraying amixture of tacrolimus in a melted hydrophilic vehicle onto the inertporous solid carrier.
 48. The method of claim 41, wherein the once-dailyextended release tacrolimus formulation releases at most 63.5% of thetacrolimus in the dosage form at the 12 hours time point, when testedaccording to USP II dissolution test (paddle) method in 900 ml of anaqueous medium at pH 4.5 and comprising 0.005% hydroxypropylcelluloseand 0.5% of the surfactant sodium lauryl sulfate, at 37° C.±0.5° C. anda paddle speed of 100 rpm.
 49. The method of claim 41, furthercomprising adjusting the dose of the extended release tacrolimus dosageform so that the whole blood pre-dose (trough) concentration oftacrolimus is maintained in the range of from about 8.8 to about 9.3ng/mL from day 2 to week 3 of tacrolimus treatment, and from about 6.5to about 8.8 ng/mL for month 1 to
 12. 50-60. (canceled)
 61. The methodof claim 41, wherein the recipient is African-American.
 62. A method ofsuppressing organ rejection in a de novo organ transplant recipient, themethod comprising (a) initiating oral treatment with a once-dailyextended release tacrolimus dosage form at an initial dose of from about0.15 to about 0.20 mg/kg/day following transplantation, wherein (i) theonce-daily extended release tacrolimus formulation provides afluctuation of less than 80% or a swing less than 120%; and (ii) theonce-daily extended release tacrolimus formulation releases thetacrolimus substantially in the colon and/or the lower ileum; and (b)concomitantly treating the recipient with an IL-2 receptor antagonistand mycophenolate mofetil.
 63. The method of claim 62, wherein therecipient is African-American.
 64. The method of claim 63, whereindosing is initiated within 48 hours following transplantation.
 65. Themethod of claim 63, wherein dosing is initiated within 24 hoursfollowing transplantation.
 66. The method of claim 63, wherein the IL-2receptor antagonist is basiliximab.
 67. The method of claim 63, wherein1 g of mycophenolate mofetil is administered twice daily (2 g dailydose).
 68. The method of claim 63, further comprising concomitantlytreating the recipient with one or more corticosteroids.